title: Phenotypic characterization of drug resistance-associated mutations in HIV-1 RT connection and RNase H domains and their correlation with thymidine analogue mutations.
abstract: OBJECTIVE
  HIV-1 reverse transcriptase (RT) mutations associated with antiviral drug resistance have been extensively characterized in the enzyme polymerase domain. Recent studies, however, have verified the involvement of the RT C-terminal domains (connection and RNase H) in drug resistance to RT inhibitors. In this work, we have characterized the correlation of recently described C-terminal domain mutations with thymidine analogue mutations (TAMs), as well as their phenotypic impact on susceptibility to zidovudine and nevirapine.
  
  
  METHODS
  HIV-1 RT sequences from Brazilian patients and from public sequence databases for which the C-terminal RT domains and treatment status were also available were retrieved and analysed for the association of C-terminal mutations and the presence of TAMs and treatment status. Several C-terminal RT mutations previously characterized were introduced by site-directed mutagenesis into an HIV-1 subtype B molecular clone in a wild-type, TAM-1 or TAM-2 pathway context. Mutants were tested for drug susceptibility to the prototypic drugs zidovudine and nevirapine.
  
  
  RESULTS
  Subtype B-infected patient database analysis showed that mutations N348I, A360V/T, T377M and D488E were found to be selected independently of TAMs, whereas mutations R358K, G359S, A371V, A400T, K451R and K512R increased in frequency with the number of TAMs in a dose-dependent fashion. Phenotypic analysis of C-terminal mutations showed that N348I, T369V and A371V conferred reduced susceptibility to zidovudine in the context of the TAM-1 and/or TAM-2 pathway, and also conferred dual resistance to nevirapine. Other mutations, such as D488E and Q547K, showed TAM-specific enhancement of resistance to zidovudine. Finally, mutation G359S displayed a zidovudine hypersusceptibility phenotype, both per se and when combined with A371V.
  
  
  CONCLUSIONS
  This study demonstrates that distinct RT C-terminal mutations can act as primary or secondary drug resistance mutations, and are associated in a complex array of phenotypes with RT polymerase domain mutations.
authors.affiliation: Departamento de Genética, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Renan B
authors.initials: RB
authors.lastname: Lengruber
authors.forename: Krista A
authors.initials: KA
authors.lastname: Delviks-Frankenberry
authors.forename: Galina N
authors.initials: GN
authors.lastname: Nikolenko
authors.forename: Jessica
authors.initials: J
authors.lastname: Baumann
authors.forename: André F
authors.initials: AF
authors.lastname: Santos
authors.forename: Vinay K
authors.initials: VK
authors.lastname: Pathak
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Soares
mesh_terms: D019943:Amino Acid Substitution; D019380:Anti-HIV Agents / Q000494:pharmacology*; D001938:Brazil; D024882:Drug Resistance, Viral; D015658:HIV Infections / Q000821:virology; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000302:isolation & purification; D006801:Humans; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics; D020125:Mutation, Missense; D019829:Nevirapine / Q000494:pharmacology; D017434:Protein Structure, Tertiary / Q000235:genetics; D013936:Thymidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015215:Zidovudine / Q000494:pharmacology
chemical_list: D019380:Anti-HIV Agents; D050505:Mutant Proteins; D015215:Zidovudine; D019829:Nevirapine; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D013936:Thymidine